KR102089449B1 - Production method of paramylon by co-cultivation of Euglena gracilis and bacterium Vibrio natriegens - Google Patents

Abstract

The present invention relates to a method of promoting the growth of euglena by co-culturing Bacterium vibrio natriegens and Euglena gracilis, and increasing the production of paramylon, a beta-glucan polymer. The method for producing paramylon according to the present invention promotes the growth of euglena gracilis and increases the production of paramylon by co-culturing Euglena gracilis and Bacterium vibrio natriegens, and thus the paramylon, the beta-glucan polymer, can be produced effectively. Particularly, in the method of the present invention, the optimal inoculation culture concentration ratio and a bacterial inoculation time is confirmed that the production of paramylon can be maximized when co-culturing Euglena gracilis and Bacterium vibrio natriegens, thereby being useful for the production of raw beta-glucan materials.

Description

Production method of paramylon by co-cultivation of Euglena gracilis and bacterium Vibrio natriegens

The present invention relates to a method of promoting the growth of euglena by co-cultivating Bacterium vibrio natrigens and euglena gracilis, and increasing the production of paramylon, a beta-glucan polymer.

Beta-1, 3-glucan (β-1, 3-glucan) is a substance known to help strengthen immunity, antioxidant activity, and control cholesterol and blood pressure levels. Thanks to these functions, it is used in various places such as cosmetics and health functional foods, and as demand increases, interest in beta-glucan production raw materials is also increasing. Beta-glucan exists in various organisms such as mushrooms, grains, and bacteria, but production of beta-glucan from them is low or difficult to extract.

Euglena is widely known as the Japanese name for "insect green" in Japan, and is a scientific name for microorganisms that inhabit fresh water such as swamps, ponds, and rice fields. Euglena is said to be a very rare microorganism belonging to the classification of both plants and animals in taxonomy because it exhibits animal characteristics such as motility by flagella or cell contraction while actively performing photosynthesis with chloroplasts. You can. Currently, various industries are paying attention to the possibility of producing useful substances by algae containing euglena. The reason why algae is dried is that commercialization as a food has already been performed in some parts, and it has the potential to produce useful materials such as petroleum-derived chemical replacement materials or fuel raw materials with a high yield compared to higher plants. Euglena accumulates paramylon as a carbohydrate in cells. Paramylon is a particle of a polymer body in which about 700 glucose is polymerized by β-1,3- bond.

A lot of research has been conducted on a method of producing new substances through co-cultivation between microorganisms or producing more specific substances desired. However, many of these attempts have not been studied with euglena species, especially in attempts to increase paramylon production.

Accordingly, the present inventor conducted research to develop a method capable of effectively increasing the production of paramylon, a beta-glucan polymer, by promoting the growth of euglena through co-cultivation between euglena gracilis and other microorganisms, and as a result, euglena gracilis and bacterium In the case of co-cultivation of Lyum vibrio natrigens, the present invention was completed by confirming that it can promote the growth of euglena gracilis and increase the production of paramylon. In particular, the optimal inoculation culture concentration ratio and bacterial inoculation time for the first time were identified to co-cultivate Euglena Gracilis and Bacterium Vibrio Natrigens to maximize paramylon production.

Korean Registered Patent No. 10-2013-0048234 Korean Patent Publication No. 10-2015-0134350 Korean Patent Publication No. 10-2018-0002772

Accordingly, it is an object of the present invention to provide a method for effectively producing paramylon (beta-glucan), a high value-added material.

Another object of the present invention is to provide a method for producing euglena gracilis with enhanced production capacity of paramylon.

In order to achieve the object of the present invention as described above,

The present invention comprises the steps of co-culturing Euglena gracilis and bacteria; And it provides a method for producing paramylon, comprising the step of obtaining the culture.

In one embodiment of the present invention, the bacteria may be Bacterium vibrio natrigens.

In one embodiment of the present invention, the euglena gracilis and bacteria may be inoculated at a concentration ratio of 1:10 and co-cultured.

In one embodiment of the present invention, the euglena gracilis may be an exponential phase cell.

In one embodiment of the present invention, the culture may include paramylon in a dry weight basis of 9.5 ~ 10 g / L.

In addition, the present invention provides a method for producing euglena gracilis in which paramylon production is enhanced, comprising the step of co-culturing euglena gracilis and bacterium vibrio natrigens.

In one embodiment of the present invention, paramylon produced by the above method may have an average granular particle size of 3 to 4 μm.

The method for producing paramylon according to the present invention promotes the growth of euglena gracilis and increases the production of paramylon by co-culturing Euglena gracilis and Bacterium vibrio natrigens. It can be produced effectively. Particularly, in the method of the present invention, the optimal inoculation culture concentration ratio and the bacterial inoculation time to confirm the optimal inoculation culture concentration that can elicit maximum production of paramylon when co-cultivating Euglena gracilis and Bacterium vibrio natrigens, beta glucan raw materials It can be useful for production.

1 is a graph showing the increase in dry weight and increase in paramylon production of euglena according to co-culture with euglena by bacteria type.
Figure 2 shows the change in the dry weight and paramylon production of euglena according to the rate of bacterial inoculation. (A: Euglena, B: Bacteria)
Figure 3 shows the change in the production of paramylon of euglena according to the timing of bacterial inoculation.
Figure 4 shows the size change of the paramylon granules according to co-culture with bacteria and euglena.
5 is a comparison of the paramylon structure according to co-culture with bacteria and euglena.

The present invention is characterized by providing a method for producing paramylon through a process of co-culturing bacteria with euglena gracilis.

'Euglena Grasilis ( Euglena) mentioned in this specification gracilis ) 'is one of the protozoa living in fresh water and can produce various useful substances such as alpha-tocopherol, a kind of vitamin, paramylon, a beta-glucan polymer, and lipid. Particularly, when sugar culture is performed under cancer culture conditions, paramylon content is more than 60% by dry weight, and cell wall does not exist, so paramylon extraction is relatively easy compared to other organisms. In addition, since the paramylon structure is composed of beta-1 and 3 bonds, it can be considered that it has a superior function than beta-glucan extracted from mushrooms and grains in which other bonds are mixed.

The term "paramylon" referred to herein is a beta-glucan polymer as an intracellular storage material of euglena.

The method for producing paramylon according to the present invention includes co-culturing Euglena gracilis and bacteria; And it may include the step of obtaining the culture.

In one embodiment of the present invention, the bacteria may be Bacterium vibrio natrigens.

In the following examples of the present invention, Pseudomonas ( Pseudomonas) is used as a type of bacteria in order to select a type of bacteria capable of co-cultivation with Euglena. sp), Vibrio natriegens ), Paenibacillus yonginensis , sphingomonas panaciterrae ) were co-cultured with Euglena, and as a result, it was confirmed that only Vibrio Natrigens can promote the growth of Euglena when co-cultured with Euglena.

In another embodiment of the present invention, the euglena gracilis and bacteria may be inoculated and co-cultured at a concentration ratio of 1:10, and the euglena gracilis may be cells in an exponential phase.

In the following examples of the present invention, the optimal inoculation culture concentration ratio and bacterial inoculation time were confirmed to maximize paramylon production when co-culturing Euglena gracilis and Bacterium vibrio natrigens, and as a result, Euglena gras Silice and bacteria showed the best paramylon production when inoculated and cultured at a concentration ratio of 1:10, and the best time to inoculate bacteria was paramylon production when euglena gracilis was an exponential phase cell. .

In the case of producing paramylon by the method of the present invention, paramylon may be included in 9.5 to 10 g / L based on the dry weight of the culture.

In addition, the present invention provides a method for producing euglena gracilis in which paramylon production is enhanced, comprising the step of co-culturing euglena gracilis and bacterium vibrio natrigens.

In one embodiment of the present invention, paramylon produced by the above method may have an average granular particle size of 3 to 4 μm.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

< Example  1>

Euglena culture and growth, Paramylon  Measure

In the present invention, Euglena gracilis ( Euglena gracilis strain Z) species were used. The above microalgae were purchased from Culture Collection of Autotrophic Organisms at the Institute of Botany of the Academy of Sciences of the Czech Republic. Modified Hutner's medium was used as the culture medium, and in 1L tertiary distilled water (unit: g) glucose, 20; glutamic acid, 5; malic acid, 5; glycine, 2.5; asparagine, 2; urea, 0.4; succinic acid, 0.1; KH ₂ PO ₄ , 0.4; MgSO _{4 ·} 7H ₂ O, 0.14; MgCO ₃ , 0.4; CaCO ₃ , 0.1; trace elements solution, 10 mL / L; Vitamin solution, 10mL / L and H ₃ BO ₃ solution, 10ml / L were added. Trace elements solution in 1L tertiary distilled water (unit: g) (NH ₄ ) ₂ Fe (SO ₄ ) _{2 ·} 6H ₂ O, 2.1; ZnSO _{4 ·} 7H ₂ O, 1.1; _{MnSO 4 · H 2 O, 0.58} ; _{(NH 4) 5 Mo 7 O} 24 · 4H 2 O, 0.18; CoSO _{4 ·} 7H ₂ O, 0.024; It was prepared by adding CuSO _{4 ·} 5H ₂ O, 0.077 and NaNO _{3 ·} 4H ₂ O, 0.074. Vitamin solution was made by adding thiamin, 0.06 g and cyanocobalamin 0.005 g to 1 L tertiary distilled water, and H ₃ BO ₃ solution was made by adding 0.029 g to 1 L tertiary distilled water. The medium was used after sterilization at 121 ° C for 20 minutes. 500 mL Erlenmeyer flask (manufactured by DURAN) was added with 250 mL of medium to perform seed cultivation, and the culture temperature was 28 ° C and stirred at 150 rpm under dark conditions.

Euglena dry weight was measured using a filter with a pore size of 1.45um.

To extract paramylon, euglena cells were rinsed twice with 99.9% ethanol, and then trypsin (1 g / L) was added to 0.1 M sodium phosphate buffer (pH 7.6) and stored at 37 ° C for more than one day. After that, the supernatant was removed by centrifugation, rinsed with a solution of urea (500 g / L), and then rinsed twice with tertiary distilled water. The extracted paramylon was dried in a drying oven for more than a day to measure weight. To measure the size of the paramylon granules, a scanning electron microscope was used. Nuclear magnetic resonance ¹³ C was used to analyze the paramylon structure.

< Example  2>

Co-culture of various types of bacteria and euglena

In this experiment, Pseudomonas ( Pseudomonas) was selected as the type of bacteria to select the type of bacteria that can be co-cultured with Euglena. sp . DSM 25356, Pseudomonas sp . DSM 25842), Vibrio natriegens KCTC 12726, Paenibacillus yonginensis KCTC 33428), sphingomonas panaciterrae KCTC 42346) each was co-cultured with Euglena. All of the above bacteria are strains known to produce a kind of phytohormone (Indole-3-acetic acid, IAA). The bacterial strains were pre-sold by the Korea Biological Resource Center (KCTC) or the German Biological Resource Center (DSMZ).

The culture medium was Marine Broth medium 2216 (BD Difco) for Vibrio Natrigens, TSA medium for Penicillus Yongin Ensis and Sphingomonas panacetera, and Nutrient Broth medium (BD Difco) for Pseudomonas culture. Was used. The bacterial culture was stirred at 28 ° C at 150 rpm. Euglena Medium was used for co-culture of Euglena, and Euglena Medium in 1L tertiary distilled water (unit: g) sodium acetate, 1; beef extract, 1; tryptone, 2; It was prepared by adding yeast extract and CaCl ₂ · H ₂ O, 0.01. For co-culture, 100 mL of Euglena Medium was added to a 150 mL Erlenmeyer flask and cultured at 150 rpm and 28 ° C. The initial inoculation concentration of Euglena was 1 × 10 ⁶ cells / mL, and the bacteria were inoculated at a ratio of 1: 1, 1:10 and 1:50 (based on the number of cells) according to the experiment.

As a result, as shown in FIG. 1, it was confirmed that only when co-culture of Euglena and Vibrio Natrigens can increase the dry weight and paramylon production of Euglena.

Through the above results, the bacteria for co-culture were selected from Vibrio natriegens species.

< Example  3>

Euglena according to Vibrio bacteria inoculation rate Dry weight  And Paramylon  Production change

According to the method of the present invention, vibrio bacteria were co-cultured in euglena cells at a ratio of 1: 1, 1:10, and 1:50.

As a result, as shown in FIG. 2, when co-cultured with bacteria (Vibrio natrigens), the dry weight and paramylon production of euglena increased, and the highest increase effect was observed when the euglena: bacterial ratio was 1:10. there was. The above results suggest that co-cultivation of Euglena and Vibrio bacteria has an effect of promoting the growth and production of metabolites.

< Example  4>

Euglena according to the timing of vibrio bacteria Paramylon  Production change

According to the method of the present invention, the bacteria were inoculated according to the growth period (oh, 72h, 120h) of the euglena with the euglena: bacteria ratio of 1:10 to find the optimal inoculation time for the bacteria, and the changes were confirmed.

As a result, as shown in FIG. 3, the production of paramylon has increased since the time of adding the bacteria (Vibrio natrigens). Particularly, when euglena was in the exponential stage, the effect of promoting paramylon production was the greatest. For reference, the exponential phase refers to a period in which the growth of protoplasm becomes maximum and constant speed in the development process of microalgae. It is the period with the shortest generation time and the most active use of metabolites for the synthesis of protoplasts.

< Example  5>

Paramylon  Shape and structure analysis

4 and 5 analyze the shape and structure of paramylon produced by Euglena. The paramylon shape was analyzed using a scanning electron microscope (S-4700, Hitachi), and the structure was analyzed using nuclear magnetic resonance ¹³ C.

As a result, as shown in FIG. 4, it was confirmed that the shape of paramylon produced by Euglena increased in size when co-cultured with bacterial bacteria (Vibrio natrigens) than in Euglena alone culture. This means that Vibrio bacteria affect the size of euglena-produced paramylon.

As a result of analyzing the paramylon structure using ¹³ C nuclear magnetic resonance, as shown in FIG. 5, it was confirmed that the structure (beta-1, 3 bond) of paramylon does not change even when co-cultivation between euglena and vibrio bacteria is performed.

Through the above results, it was confirmed that co-cultivation increases the production of paramylon while not affecting the structure of paramylon, so there is no problem in commercial use.

So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

Claims (7)

A method for producing paramylon, comprising the step of obtaining a culture co-cultured with Euglena gracilis and Bacterium vibrio natrigens. delete According to claim 1,
The euglena gracilis and bacteria are inoculated at a concentration ratio of 1:10 and co-cultured, characterized in that the production method of paramylon. According to claim 1,
The euglena gracilis is a method of producing paramylon, characterized in that the cells of the exponential phase (exponential phase). According to claim 1,
The culture method for producing paramylon, characterized in that it comprises paramylon in a concentration of 5 ~ 6g / L. A method for producing euglena gracillis with enhanced production of paramylon, comprising the step of co-culturing euglena gracilis and bacterium vibrio natrigens. The method of claim 6,
Paramylon produced by the above method is characterized in that the average granular particle size is 3 ~ 4μm, euglena gracilis production method with enhanced paramylon production.

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